How large can be cloned into pUC19 ?

Phillip San Miguel pmiguel at bilbo.bio.purdue.edu
Tue Dec 30 12:05:17 EST 1997



Linchengtao wrote:

> Hi,netters:
>   Wish a good new year to everybody!
>   And is there anyone who have the experiences of cloning large
> fragment into pUC19 ? I wonder the biggest size that can be cloned into pUC19 but not quite influence pUC19's high copy character in host(Ecoli). Thank you!
>
> linchengtao

    A 14 kb EcoRI/KpnI chunk of the Erwinia stewartii carotenoid operon in pUC19 seems very high copy number.  That is, my yields from plasmid preps seem good.
But would you really notice if the copy number dropped to 1/2 or even 1/5th the non recombinant pUC copy number?   Usually for an insert below 15 kb or so I
don't expect a drastic reduction in the yield of a subclone in a high number vector (e.g., pBluescript[and its 2 kb derivative, pMOB], pGEM or  pUC.)  When my
inserts are larger than 18-20 kb I will frequently notice a decline in my DNA yield from a miniprep.
    The largest insert into a high copy number vector I've heard of was by a guy in our lab, Alex Tikhonov.  He subcloned a 32 kb NotI fragment from a genomic
sorghum BAC into pBluescript and pMOB.  This subclone sometimes refused to grow.  Note that if this subclone were kept at the normal pBluescript copy number
(~500?) it would be forcing the bacterial host to replicate many times more plasmid DNA than is contained in its own chromosome.
    I wish there were a strain that would maintain the copy number of very large plasmids -- even if it grew much slower than a normal strain.  Sometimes I get
a good yield of cells but a poor yield of plasmid.  The strains we usually use are DH5alpha and XL1Blue.  Sometimes when I am growing big plasmids I grow the
cultures at 30 oC and hope that in growing slower they might keep the plasmid copy number up.  I get variable results from this technique so it may not help at
all.
   Another problem may be the use of beta-lactamase encoding plasmids (Amp resistant).  Ampicillin is thermo-labile and beta-lactamase is secreted into the
media by the host strains.  If I were using kanamycin or another antibiotic there might be less problem.  There is a version of pBluescript that encodes
chloramphenicol resistance.

Phillip San Miguel




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